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SUMMARYAs population genomics is transitioning from single reference genomes to pangenomes, major improvements in terms of genome contiguity, phylogenetic sampling, haplotype phasing and structural variant (SV) calling are required. Here, we generated theSaccharomyces cerevisiaeReference Assembly Panel (ScRAP) comprising 142 reference-quality genomes from strains of various geographic and ecological origins that faithfully represent the genomic diversity and complexity of the species. The ca. 4,800 non-redundant SVs we identified impact the expression of genes near the breakpoints and contribute to gene repertoire evolution through disruptions, duplications, fusions and horizontal transfers. We discovered frequent cases of complex aneuploidies, preferentially involving large chromosomes that underwent large SVs. We also characterized the evolutionary dynamics of complex genomic regions that classically remain unassembled in short read-based projects, including the 5 Ty families and the 32 individual telomeres. Overall, the ScRAP represents a crucial step towards establishing a high-quality, unified and complete S. cerevisiae pangenome.

Polyploidization events are observed across the tree of life and occur in many fungi, plant, and animal species. During evolution, polyploidy is thought to be an important source of speciation and tumorigenesis. However, the origin of polyploid populations is not always clear, and little is known about the precise nature and structure of their complex genome. Using a long-read sequencing strategy, we sequenced 71 strains from the Brettanomyces bruxellensis yeast species, which is found in anthropized environments (e.g., beer, contaminant of wine, kombucha, and ethanol production) and characterized by several polyploid subpopulations. To reconstruct the polyploid genomes, we phased them by using different strategies and found that each subpopulation had a unique polyploidization history with distinct trajectories. The polyploid genomes contain either genetically closely related (with a genetic divergence 3%), indicating auto- as well as allopolyploidization events. These latest events have occurred independently with a specific and unique donor in each of the polyploid subpopulations and exclude the known Brettanomyces sister species as possible donors. Finally, loss of heterozygosity events has shaped the structure of these polyploid genomes and underline their dynamics. Overall, our study highlights the multiplicity of the trajectories leading to polyploid genomes within the same species.

Phasing, and in particular polyploid phasing, have been challenging problems held back by the limited read length of high-throughput short read sequencing methods which can't overcome the distance between heterozygous sites and labor high cost of alternative methods such as the physical separation of chromosomes for example. Recently developed single molecule long-read sequencing methods provide much longer reads which overcome this previous limitation. Here we review the alignment-based methods of polyploid phasing that rely on four main strategies: population inference methods, which leverage the genetic information of several individuals to phase a sample; objective function minimization methods, which minimize a function such as the Minimum Error Correction (MEC); graph partitioning methods, which represent the read data as a graph and split it into k haplotype subgraphs; cluster building methods, which iteratively grow clusters of similar reads into a final set of clusters that represent the haplotypes. We discuss the advantages and limitations of these methods and the metrics used to assess their performance, proposing that accuracy and contiguity are the most meaningful metrics. Finally, we propose the field of alignment-based polyploid phasing would greatly benefit from the use of a well-designed benchmarking dataset with appropriate evaluation metrics. We consider that there are still significant improvements which can be achieved to obtain more accurate and contiguous polyploid phasing results which reflect the complexity of polyploid genome architectures.

Yeasts, and in particular Saccharomyces cerevisiae, have been used for brewing beer for thousands of years. Population genomic surveys highlighted that beer yeasts are polyphyletic, with the emergence of different domesticated subpopulations characterized by high genetic diversity and ploidy level. However, the different origins of these subpopulations are still unclear as reconstruction of polyploid genomes is required. To gain better insight into the differential evolutionary trajectories, we sequenced the genomes of 35 Saccharomyces cerevisiae isolates coming from different beer-brewing clades, using a long-read sequencing strategy. By phasing the genomes and using a windowed approach, we identified three main beer subpopulations based on allelic content (European dominant, Asian dominant, and African beer). They were derived from different admixtures between populations and are characterized by distinctive genomic patterns. By comparing the fully phased genes, the most diverse in our dataset are enriched for functions relevant to the brewing environment such as carbon metabolism, oxidoreduction, and cell wall organization activity. Finally, independent domestication, evolution, and adaptation events across subpopulations were also highlighted by investigating specific genes previously linked to the brewing process. Altogether, our analysis based on phased polyploid genomes has led to new insight into the contrasting evolutionary history of beer isolates.

While genome sequencing and assembly are now routine, we still do not have a full and precise picture of polyploid genomes. Phasing these genomes, i.e. deducing haplotypes from genomic data, remains a challenge. Despite numerous attempts, no existing polyploid phasing method provides accurate and contiguous haplotype predictions. To address this need, we developed nPhase, a ploidy agnostic pipeline and algorithm that leverage the accuracy of short reads and the length of long reads to solve reference alignment-based phasing for samples of unspecified ploidy (https://github.com/nPhasePipeline/nPhase). nPhase was validated on virtually constructed polyploid genomes of the model species Saccharomyces cerevisiae, generated by combining sequencing data of homozygous isolates. nPhase obtained on average >95% accuracy and a contiguous 1.25 haplotigs per haplotype to cover >90% of each chromosome (heterozygosity rate ≥0.5%). This new phasing method opens the door to explore polyploid genomes through applications such as population genomics and hybrid studies.

Ale brewing yeast are the result of admixture between diverse strains ofSaccharomyces cerevisiae, resulting in a heterozygous tetraploid that has since undergone numerous genomic rearrangements. As a result, comparisons between the genomes of modern related ale brewing strains show both extensive aneuploidy and mitotic recombination that has resulted in a loss of intragenomic diversity. Similar patterns of intraspecific admixture and subsequent selection for one haplotype have been seen in many domesticated crops, potentially reflecting a general pattern of domestication syndrome between these systems. We set out to explore the evolution of the ale brewing yeast, to understand both polyploid evolution and the process of domestication in the ecologically relevant environment of the brewery. Utilizing a common brewery practice known as ‘repitching’, in which yeasts are reused over multiple beer fermentations, we generated population time courses from multiple breweries utilizing similar strains of ale yeast. Applying whole-genome sequencing to the time courses, we have found that the same structural variations in the form of aneuploidy and mitotic recombination of particular chromosomes reproducibly rise to detectable frequency during adaptation to brewing conditions across multiple related strains in different breweries. Our results demonstrate that domestication of ale strains is an ongoing process and will likely continue to occur as modern brewing practices develop.